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The effect of 1 MHz ultrasound on the proliferation of synchronized chinese hamster V-79 cells
Ultrasound in Med. & Biol., Vol.7, pp. 175-184, 1981 Printed in Great Britain.
THE
0301-56291811020175-10502.0010 PergamonPress Ltd.
EFFECT OF 1 MHz ULTRASOUND ON THE PROLIFERATION OF SYNCHRONIZED C H I N E S E H A M S T E R V-79 C E L L S
VICTOR CIARAVINO and MORTON W. MILLER Department of Radiation Biology and Biophysics, School of Medicine and Dentistry, The University of Rochester, Rochester, NY 14642, U.S.A.
and GARY E. KAUFMAN Department of Radiology and the Franklin McLean Memorial Research Institute, The University of Chicago, Chicago, IL 60637, U.S.A. (First received 22 April 1980; and in final form 30 July 1980)
AbstraetmMitotically synchronized Chinese hamster V-79 cells were exposed to continuous wave l MHz ultrasound at a spatial peak intensity of 3 W/cm 2 at 37°C. A fraction of the cells was lysed immediately, and a second fraction was rendered nonviable. Many of these intact but nonviable cells appeared to be morphologically damaged. The remaining viable cells formed smaller colonies than did unexposed controls, apparently because of the death of one daughter cell at or shortly after the first division. There were no apparent effects on cells surviving beyond that period, and progression through the cell cycle was unperturbed. Key words: Ultrasound, Cells, Cell cycle, Membrane, Survival.
INTRODUCTION
Exposure of cultured mammalian cells to ultrasound at frequencies and intensities similar to those used in medical therapy results in the lysis of a fraction of the cell population (Clarke and Hill, 1970; Fu et al., 1980; Kaufman et al., 1977). In addition, many of the remaining intact cells are rendered nonviable as determined by vital dye exclusion and colony-forming ability (Kaufman et al., 1977; Li et al., 1977). Sonicated cells grow more slowly in monolayer culture (Kaufman and Miller, 1978) and form smaller colonies (Miller et al., 1977) than do unirradiated controls. A major site for ultrasound damage appears to be the cell membrane (Williams, 1973; Watmough et al., 1977). In the present study we examine the relationship between visible morphological damage visible by phase contrast microscopy and cell death in sonicated cells. The biological mechanisms responsible for decreased growth rate in sonicated cells are also examined. Kaufman and Miller (1978) showed that at least part of the" decreased growth rate was the result of non-viable cells being sloughed off and lost from the monolayer. However, to account for formation of smaller-than-normal colonies (Miller et al., 1977), part of the cell loss must
occur at or after the first post-sonication division, and/or there must be an increase in the duration of the cell cycle. Using synchronized cells, we examine both of these possible mechanisms. METHODS o r PROCEDURE
Cell line, medium and growth conditions A Chinese hamster V-79 cell line, originally established by Dr. M. M. Elkind, was obtained from Dr. L. J. Tolmach's laboratory (Washington University, St. Louis, MO). The cells were maintained at 37°C in a 5% CO2 water-saturated environment and grown in F-10 Ham medium supplemented with 10% calf, 10% fetal calf sera, and an antibiotic (kanamycin, 100/zg/ml). Harvesting of mitotic cells and sonication The cell population used in each experiment w/Is obtained from asynchronous, exponentially growing ,gultures grown in 490cm 2 polystyrene tissue culture roller flasks (Corning Glass Works, Corning, New York) rotating at 0.3 rpm 2-3 days before use. Cells in the mitotic phase of the cell cycle were accumulated by the addition of 0.06/xg/ml colcemid (Gibco, Grand Island, New York) for 1 hr. At that time, the cells were selected by gentle shaking of the roller
175
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V. CIARAVINO et al.
bottle, a modified method of Highfield and Dewey (1975). The cells were then centrifuged and resuspended in fresh medium to remove the colcemid. The mitotic index of the cells at the onset of each experiment was determined to be 90-95%, which is in agreement with Highfield and Dewey (1975). Three ml aliquots of suspended cells ( 105cells/ml) were exposed to ultrasound in polystyrene tubes as described by Kaufman and Miller (1978). A l/8in, steel ball radiometer shows that the tubes are exposed to a typical bell-shaped beam with approximately a 35% attenuation of the sound field due to the front half of the polystyrene tube. Fifty percent is approximately 3 mm off center. Within the tube there appear to be standing waves as evidence by points in the tube where a steel bali radiometer shows almost no movement with changes in the power to the transducer. Elsewhere in the tube the steel ball appears to get caught in streaming eddies and exhibits an orbital motion. Hence, the physical situation within the tube is quite complex. All exposures were continuous wave (CW) at a frequency of 1.07 MHz, and at a spatial peak intensity of 3.0 W/cm 2 (37°C) as determined by a steel ball radiometer (Dunn et al., 1977). All control populations were sham-exposed.
durations of l, 2, 5 and l0 rain. After sham and ultrasound exposures, the cells were counted on a Coulter Counter Model B (Coulter Electronics, Hialeah, Florida). The percentage of intact cells was determined and aliquots of cells (200-300 cells for control plates, - 2 - 3 x l03 cells for "sonicated" plates) were placed in 100 mm diameter Petri dishes (Falcon, Oxnard, CA). Dishes were then coded before scoring. At various time intervals a dish was removed from the incubator and scored for the number of cells per colony, 100 colonies per dish were scored. For the initial time interval (2 hr), the data were derived from potential colonies which had either one or two cells (i.e. l < average value<2). The plates were then replaced in the incubator and the procedure repeated at various intervals for a period of 54 hr. There were four plates each for control and sonicated cells; in this manner one particular plate was scored a maximum of three times in the 54 hr period of any one experiment. This alleviated the problem of using one plate and repeatedly scoring the colonies, thereby decreasing the chance of cells being detached from the monolayer. The experiment was conducted three times for each exposure duration.
Daughter cell survival Cell growth After treatment, the control and sonicated cell suspensions were counted on a Coulter Counter and the cells were placed into six 25 cm 2 culture flasks (Falcon, Oxnard, CA), three of which were controls ( - 1 . 5 x 105 cells/flask). At l, 2 and 3 hr intervals after sonication, a control and a "sonicated" flask were removed from the incubator, the cells detached by agitation and the cell number was again determined. The fraction of viable cells was determined by the ratio of the number of cells excluding trypan blue in 0.4% aqueous solution to the total cell number (Phillips, 1973); one hundred cells per slide were scored. The experiment was repeated four times; all slides were scored blind. Preliminary data on cells fixed in 3:1 methanol:acetic acid or flame-killed cells indicated 100% non-viable cells with the trypan blue staining procedure.
Colony multiplicity Mitotic cells were exposed to 1MHz ultrasound at an intensity of 3 W/cm 2 for
The possibility that one of the two daughter cells may be lost at or shortly after division was tested. After sonication or sham sonication at 3 W/cm 2 for 1 min, mitotic cells were pipetted into 125 ml Erhlenmeyer flasks containing conditioned medium (conditioned medium contains equal amounts of fresh medium and medium previously used to support cell growth) and placed on a shaker at 37°C for 90 min. Carrano (1973) states that cells are almost completely released from mitosis (or more correctly, from metaphase arrest) within 45 min after conditioned medium is added. The cell suspensions were then placed on 100mm Petri dishes and scored for colony multiplicity as previously described. The experiments were repeated four times.
Incidence of polyploidy Mitotic cells were exposed to 3W/cm 2 ultrasound for 1 min. The sham and treated cells were placed on 100 mm Petri dishes and 5 mls of fresh medium added, 3 dishes each for sham and treated cells. The dishes were
The effect of 1 MHz ultrasound on the Chinese hamster V-79 cells
then placed in the incubator at 37°C. At 24 hr post-sonication 0.30 ml of colcemid (0.06 tzg/ml) was added to two of the dishes, one control and one "sonicated," for 4 hr to accumulate mitotic figures. The mitotic cells were then collected, given a 10 min treatment in hypotonic solution (1:1 F-10 medium:distilled water), and fixed in 3:1 methanol:acetic acid. Slides were prepared and Giemsa stained. The same procedure was repeated at 46 and 72 hr after sonication. The slides were then coded and the percent of cells with polyploid mitotic figures determined. The experiments were scored blind and repeated 4 times.
Autoradiographical analysis of progress into the S phase of the cell cycle after sonication Mitotically synchronized cells were exposed to 3 W/cm 2 ultrasound for 1 min. The cells were placed onto 35 mm Petri dishes and 2 ml of fresh medium containing tritiated thymidine (1.0~Ci/mM) were added. The dishes were then placed in an incubator at 37°C. At hourly intervals duplicate dishes of sham and ultrasound treated cells were removed from the incubator and fixed with 3:1 methanol:glacial acetic acid. The dishes were then treated with 2% perchloric acid (W/V) overnight in a refrigerator, rinsed with distilled water and with 95% ethanol and air dried. NTB-3 photographic emulsion (Eastman Kodak, Rochester, New York) at 45°C was poured into the dishes. The excess emulsion was poured off, and the dishes were inverted to dry for several hours. The plates were then stored in a light-tight box in the refrigerator for 1-2 weeks, and developed with D-19 (Eastman Kodak, Rochester, New York) for 2 min at 19°C. After fixation, the dishes were Giemsa stained and coded before scoring. Dishes were scored for percent 3Hnuclear labeled cells (Quastler and Sherman, 1959).
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(1.0ttC/ml). After 30min the dishes were removed from the incubator and treated in the same manner as described in the above section. Development of autoradiographical dishes and scoring was as described above. The cell cycle was also assessed by determining the frequency of cells in mitosis after release from synchrony. Approximately 104 cells were placed onto 25cm 2 Falcon flasks and 5 ml of conditioned medium added after a 1 min sonication at 3 W/cm 2. At 6, 12, 14, 16, 18 and 20 hr post-sonication the cells were analyzed for the percent mitotic cells in the population. One hour before a designated time, 0.30 ml colcemid (0.06mg/ml; Gibco, Grand Island, New York) was placed into the flasks. After one hour the medium was removed and placed in a test tube and spun down. At that time 5 ml of 0.5% sodium citrate were placed both on the remaining attached cells and on the cell pellet in the test tube. After 30 min the cells were detached by vigorous shaking of the flask; the contents of the flask and the tube were combined, spun down and fixed in 3:1 methanol:glacial acetic acid. Slides were prepared and scored as described previously. The cell cycle experiments were repeated four times.
Cell survival
The plates used in the colony multiplicity experiments were kept in a growth incubator for 7 days, then fixed in 3:1 methanol:glacial acetic acid for 5 min, and Giemsa stained. The number of colonies on the plates was counted using a dissecting microscope. Colonies which contained fewer than fifty cells were not included in the total number of colonies. The plating efficiency of cells was determined as the number of colonies per plate divided by the number of intact cells plated. The surviving fraction was calculated by dividing the plating efficiency of sonicated cells by the plating efficiency of sham treated Analysis of progression through the 1st cell cells. The experiments were repeated four times.
cycle after sonication
Mitotically synchronized cells were exposed to 3 W/cm 2 ultrasound for 1 min. Progress through the cell cycle immediately following sonication was assessed in two ways. Cells were placed onto 35 mm Petri dishes and at 2 hr intervals dishes containing control and sonicated cells were removed from the incubator, the medium was aspirated and replaced with medium containing 3H-TdR
Analysis of morphological perturbation Within 15 min after sonication at an intensity of 3W/cm 2 for 0, 1, 2, 5 or 10min cells were plated onto clean microscope slides and viewed unstained, through a Zeiss Photomicroscope (phase contrast, 80x magnification). Slides were scored for percent aberrant cells, that is, cells which appeared morphologically damaged.
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V. CIARAVINO et al. RESULTS
Initial cell lysis Exposure of mitotically synchronized Chinese hamster V-79 cells to 1 MHz ultrasound at an intensity of 3 W/cm 2 resulted in the immediate lysis of a fraction of the cell population. As shown in Fig. 1, the amount of cell lysis was directly correlated to the time during which cells remained in the sound field. Temperature rises do not appear to be involved in the cellular changes reported since the cells are maintained at 37°C before, during and after sonication. For a 1.0min exposure there were 83.56+_1.92 (S.E.)% cells remaining intact. This percentage decreased to 53.06-+6.71% for a 2min exposure, 39.98+-4.30% for a 5 min exposure and 7.63+-1.04% for a 10min exposure.
Cell growth The changes in cell number and viability during the first 3 hr after sonication for 1 min at 3 W/cm 2 are given in Table 1. The growth index in column 1 is the ratio of the number of cells in the flask at the indicated time to
100 : - -
--~ ~ 10
g
~ 5! (..) ~,
~
z
o
FREQUENCY 1 MHz I N T E N S I T Y 3W/cm 2 (~MEAN ± S E
~
~
~
the number of intact cells initially plated. Column 2 indicates the number of non-viable cells as determined by trypan blue dye exclusion. The growth index is then corrected for non-viable cells (column 3) by multiplying the growth index (column 1) by (1.00-nonviable cell index). The number of cells in the control flasks increased approximately 60% in the first hour as the cells divided, then remained steady for the next 2 hr. The fraction of non-viable cells was approximately 15% and remained about constant. The number of sonicated cells (and their daughters) likewise increased in the first hour by approximately the same percentage as controls, but approximately twice as many sonicated cells (i.e. - 3 0 % ) were non-viable. During the next 2hr there was a decrease in cell number, indicating some cell lysis, and an increase in the fraction of cells unable to exclude the trypan blue, indicating an increase in the number of non-viable cells detected.
Colony multiplicity Colony multiplicity data are shown in Fig.2 for an exposure duration of 1 min, and given in Table 2 for exposure durations of up to 10min. Colonies which were derived from sonicated cells were smaller (i.e. contained fewer cells) than controls. This difference appeared within the first few hours after sonication. Thereafter growth rates were comparable for controls and sonicated cells.
Daughter cell survival ~
l~
E X P O S U R E D U R A T I O N (minutes)
Fig. 1. The relationship between sonication duration and percent intact cells at an exposure of 3W/cm 2. Each point in this figure presents the mean-+S.E, of three experiments. Cell counts were determined in triplicate.
In these experiments the cultures were maintained in suspension for 90 min after sonication or sham-sonication to allow the first division to be completed before the cells were plated. Thus the colonies were started from single daughter cells. There was no
Table 1. The growth of V-79 cells after exposure to 3 W/cm 2 for 1 min. The corrected growth index of sonicated cells has been normalized to their respective controls. (Refer to Results, Cell growth)
Control
Hr. a f t e r Sonication
Growth Index
Non-Viable Cell Index
Growth Index Corrected f o r Non-Viable Cells
1 2 3
1.57 1.65 1.52
O. 14 0.16 0.13
i . 35 1.39 1.32 Normalized Values
Sonicated
i 2 3
1.56 i . 43 1.38
0.29 fh 34 0.42
1.11 O. 94 0.80
0.82 O. 68 0.59
The effect of 1 MHz ultrasound on the Chinese hamster V-79 cells
179
Table 2. Colony Multiplicity of Chinese Hamster V-79 Cells After Exposure to 3 W/cm 2 Ultrasound Exposure Duration (min)
Time After Exposure (hrs)
Colony M u l t i p l i c i t y (no. cells/colony)
Exposed
i
2
5
i0
Control
1 2 3 4 6 12 24 30 48 54
1.07 1.28 1.26 1.22 1.21 1.50 2.33 2.32 5.44 7.62
1.27 1.46 1.42 1.47 1.50 1.71 2.67 2.85 6.72 9.59
.84 .88 .89 .83 .81 .88 .87 .81 .81 .79
2 3 6 12 24 30 48 54
1.08 1.31 1.20 1.36 i . 87 2.07 3.35 4.85
i . 19 1.51 1.47 1.65 2.35 2.72 4.28 6.96
.91 .87 .82 .82 .80 .76 .76 .70
2 6 24 30 48 54
1.16 ].27 i . 86 2.20 3.70 5.66
1.27 1.54 2.46 3.05 5.25 7.12
.ql .82 .76 .72 .70 .79
2 6 24 30 48 54
i.i0 1.15 2.37 2.71 4.73 5.77
1.31 1.49 2.98 3.22 5.98 8.15
.84 .77 .80 .84 .79 .71
Analysis o[ progression
l0
..~
Ratio Exposed/Control
FREQUENCY 1 MHz INTENSITY 3 W/cm2/1 min
HOURS POST-SONtCAT~ON Fig. 2. The formation of colonies with time from single cells after sonication at 3 W/cm ~ for 1 rain (0) or sham sonication (©). Each point in the figures represents the mean for three experiments, with 100 colonies scored in each experiment.
The progression of cells through G~ and into the first S period after sonication is shown in Fig. 3. The progression of cells through the first cell cycle after sonication as shown by pulse 3H-labeling is depicted in Fig. 4. Figure 5 illustrates the mitotic index of cells for the 20hr period after sonication. There were no statistically significant differences between exposed and control populations in any of these three sets of 1oo 80. ~
60
m 40
DUM~ONs E1 rnln
subsequent difference detected in colony multiplicity between colonies derived from sonicated and sham-sonicated cells. HRS. POST-SONICATION
Incidence of polyploidy Of the 1800 cells scored there was only one polypoid observed and it appeared in a slide from sham treated cells. Thus neither the colcemid treatment nor the sonication caused a significant number of polyploid cells.
Fig. 3. The relationship between the percent 3H-labeled cells and hours post-sonication. After sonication cells were placed in continuous 3H-TdR (1.0 #Ci/mi) label. At hourly intervals plates were rinsed, fixed and the plates scored for percent 3H-labeled cells. Each point represents the mean _+S.E. of three experiments. Duplicate plates were used in each experiment (O-control, O-sonicated).
V. C1ARAVINO el al.
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reduction (to - 2 . 5 % , or - 8 % of the control value).
Analysis of morphological perturbations Normal and aberrant-type mitotic cells are shown in Figs. 6(a, b). Figure 6(a) is a normal mitotic cell, well rounded and with a clearly 3 ] ~ 6 8 ; ,: L~ 8 2C defined cell membrane. In Fig. 6(b), the cell H O U R S POST SONICATION type illustrated is a mitotic cell but is Fig. 4. 3H-labeled cells as a function of time after soni- easily identified as an aberrant cell. The cation. Exposure of mitotic cells was at 3 W/cm2 for membrane is obviously damaged and l min. The experiments were repeated 4 times (O-concytoplasm appears to be emanating from the trol, Q-sonicated). cell; this process is characteristic of cell lysis. ~oo Approximately 21% of the control cells IMHZ were aberrant as determined by the above ~U~ATION I MIN criteria. A 1 min exposure to ultrasound increases the fraction of aberrant cells to 39%, and a 10 min exposure to 70%. An aberrant frequency corrected for the control ~ 20 frequency of aberrants (i.e. the increase in aberrant frequency from controls divided by 0 6 16 20 HOURS POST SONICATION the control fraction of nonaberrants) is given Fig. 5. Percent mitotic cells as a function of hours post in Table 3. This corrected f r e q u e n c y of abersinication. Exposures were at 3W/cm2 for 1 min. The rants is approximately 20% after a 1 min experiments were repeated 4 times (A-control, O-soniexposure to ultrasound, and increases to apcated). proximately 62% after a 10 min exposure. 1)
FREQUENCY
(~)
I MEAN . S E
12
~4
18
progression experiments, as determined by paired t tests and by analysis of covariance (p>0.05). Both the control and sonicated cells demonstrate a mitotic peak at 18 hr postultrasound treatment.
Plating efficiency The plating efficiency of control mitotic cells which were treated for l hr with 0.06 tzg/ml colcemid averaged - 3 0 % . As indicated in Table 3, a 1 min exposure to ultrasound reduced this plating efficiency by approximately 66% (to - 1 0 % ) , while a 5 or 10min exposure resulted in about a 92%
DISCUSSION
Exposure of synchronous Chinese hamster V-79 cells to 1 MHz ultrasound for 1-10 min at an intensity of 3 W / c m 2 resulted in the immediate lysis of a fraction of the cell population (Fig. 1). Microscopic examination of the remaining cells in the first few minutes after exposure revealed that many were morphologically damaged and appeared to be in the process of losing their physical integrity; presumably most, if not all, of these damaged cells subsequently lysed. These results are consistent with previous studies. Williams (1973) exposed mouse Ehrlich
Table 3. Platingefficiencyand aberrant cells after exposure to 3 W/cm2ultrasound. Values given are mean _+ standard error of the mean Duration of Exposure (min) i 2 5 10
•
Reduction in Plating Efficiency *(%) 66 • R6 ~ 92 i 91 *
3 4 3 4
Increase in Frequency of Aberrants f(%) 22.5 36.0 50.8 62.3
• ~ * *
2.6 2.3 6.4 I0.I
I - ( P / P o ) , where P is p l a t i n g e f f i c i e n c y a f t e r a given exposure, and Po is p l a t i n g e f f i c i e n c y f o r c o n t r o l s . Based on three experiments, four plates per experiment at each exposure d u r a t i o n .
(X-Xo)/(1-Xo), where X is the frequency of aberrant cells after a given exposure, and Xo is the frequency of aberrants for controls. Based on four experiments, 100 cells per experiment at each exposure duration.
The effect of 1 MHz ultrasound on the Chinese hamster V-79 cells
la)
(b)
Fig. 6. Photomicrographs of mitotic cells within 15 min after sonication or sham-sonication. Figure 6(a) shows a normal miotic cell. Figure 6(b) shows an aberrant cell. Photomicrographs were taken on a Zeiss photomicroscope at an 80X magnification using Kodak high contrast copy film. ( - 900X).
181
The effect of 1 MHz ultrasound on the Chinese hamster V-79 cells ascites carcinoma cells to a small-scale acoustic microstreaming field generated around a transversely oscillating wire driven at 20 kHz, and observed cells with large areas of plasma membrane torn off, but with the cytoplasm and organelles still attached to each other. Watmough et al. (1977) conducted electron microscopic examinations of HeLa cells after treatment with 0.75 MHz ultrasound at spatial peak intensities of 1W/cm ~. Damage was observed to the nuclear and mitochondrial membrane systems as well as to the plasmalemmal and endoplasmic reticulum. The morphologically damaged cells account for a significant fraction, but not all, of the loss in viability in those sonicated cells which are not lysed immediately (Table 3). This result is not surprising, since Kaufman and Miller (1978) observed that some cells rendered nonviable by ultrasound can remain floating in the medium for several days without lysing. The overall loss of viability observed in the present study (including morphologically damaged cells and those not so damaged) is consistent with that reported previously (Kaufman and Miller, 1978; Fu et al., 1980). As might be expected, with longer exposures the morphological aberrants account for a larger fraction of the nonviable cells, reflecting an overall increase in damage to the cell population. The effect of ultrasound on cell growth was examined by measuring changes in total cell number (or number of viable cells as determined by vital dye exclusion), and by measuring the average number of cells per colony (colony multiplicity). Both types of experiments indicated a reduction in growth in the sonicated cells. These results are similar to those reported previously for asynchronous cells (Miller et al., 1977; Kaufman and Miller, 1978). As described in the introduction, two mechanisms could account for this decreased growth--an increase in the duration of the cell cycle, and/or a loss of cells from the proliferating population. Progression through the cell cycle was assessed by chronic and pulse labelling experiments (Figs. 3 and 4, respectively) and by mitotic index determinations (Fig. 5). No difference between exposed and control cells was observed with respect to progression. Hence there was little or no effect on the duration of the cell cycle.
183
However, both the growth experiments (i.e. change in total cell number) and the colony multiplicity experiments indicated a loss of cells from the proliferating population. The growth experiments (Table 1) indicate that after a 1 min exposure most of the cells which were not immediately lysed proceeded through the first division and were still intact 1 hr after exposure, although a number of the daughter cells were nonviable as determined by trypan blue exclusion. During the next 2 hr there was a decrease in cell number and a • lecrease in the fraction of cells able to exclude the vital dye. Thus in the period from 1-3 hr after exposure some daughter cells were lysed, while other lethally damaged daughter cells lost the ability to exclude trypan blue. The colony multiplicity experiments give a similar picture of cell loss within the first few hours after exposure, although the effects appear more pronounced within the first hour after exposure (Fig. 2 and Table 2). This may mean that many of the intact but lethally damaged daughter cells fail to reattach to the plates after division. There appears to be a slight drop in colony multiplicity between 2 and 6 hr after sonication, for cells sonicated for 1 min, which may represent a second population of lethally damaged daughter cells which initially reattach after division, but are later sloughed off into the medium or lyse. This would be similar to the results of Kaufman and Miller (1978), who observed that, in asynchronous V-79 cells, sonication resulted in a large population of nonviable cells which initially reattached but were later sloughed off. However, the changes in colony multiplicity during this period are small and may not be significant. Beyond the first several hours after exposure there were no apparent effects on cell growth, with the ratio of colony multiplicity between sonicated and control cells remaining nearly constant. Hence the effects of ultrasound on colony size appear to be due entirely to colonies in which one daughter cell was lost at or shortly after division, while the effects on growth (total cell number) would also reflect colonies which were never formed since both daughters were lost. In the case of asynchronous cells there may also be significant loss of cells prior to the first division, which would contribute to effects on growth (Kaufman and Miller, 1978) but not to decreased colony size (Miller et al., 1977).
184
V. CIARAVINOet al.
Acknowledgements--The authors are indebted to Ms. Valarie Ferro for excellent technical assistance during the course of this research. This paper is based on work performed under contract no. DE-AC02-76EV03490 with the U.S. Department of Energy at The University of Rochester, Department of Radiation Biology and Biophysics and has been assigned Report No. UR-3490-1777. This work was also partially supported by USPHS Grants GM22680, FD00694 and CA25785. REFERENCES
Carrano, A. V. (1973) Chromosome aberrations and radiation-induced cell death. I. Transmission and survival parameters of aberrations. Mutation Research 17,341-353. Clarke, P. R. and Hill, C. (1970) Physical and chemical aspects of ultrasonic disruption of cells. J. Acoust. Soc. Am. 50, 649. Dunn, F., Auerbach, A. J. and O'Brien, Jr., W. D. (1977). A primary method for the determination of ultrasonic intensity with elastic sphere radiometer. Acustica 38, 58-61, Fu, Y-K., Miller, M. W., Kaufman, G. E., Lange, C. S. and Grifliths, T. D. (1980) Ultrasound lethality to synchronous and asynchronous Chinese hamster V-79 cells. Ultrasound in Med. and Biol 6, 39-46. Highfield, D. P. and Dewey, W. C. (1975) Use of mitotic selection procedure for cell cycle analysis: Emphasis on radiation-induced mitotic delay. In Methods in Cell
Biology (Edited by Prescott, D. M.), Vol. IX, pp. 85-101. Academic Press, New York. Kaufman, G. E. and Miller, M. W. (1978) Growth retardation in Chinese hamster V-79 cells exposed to l MHz ultrasound. Ultrasound in Med. and Biol. 4, 139-144. Kaufman, G. E., Miller, M. W., Grittiths, T. D. and Ciaravino, V. (1977) Lysis and viability of cultured mammalian cells exposed to l MHz ultrasound. Ultrasound Med. Biol. 3, 21-25. Li, G. C., Hahn, G. M. and Tolmach, L. T. (1977) Cellular inactivation by ultrasound. Nature 267, 163165. Miller, M. W., Ciaravino, V. and Kaufman, G. E. (1977) Colony size and giant cell formation from mammalian cells exposed to 1 MHz ultrasound. Radiat. Res. 71, 628-634. Phillips, H. J. (1973) Dye exclusion tests for cell viability, In Tissue Culture, Methods and Applications (Edited by Kruse, F. F. and Patterson, M. K.), pp. 406-408. Academic Press, New York. Quastler, H. and Sherman, F. G. (1959) Cell population kinetics in the intestinal epithelium of the mouse. Exptl. Cell Res. 17, 420-438. Watmough, D. J., Dendy, P. P., Eastwood, L. M., Gregory, D. W. and Gordon, F. C. A. (1977) The biophysical effects of therapeutic ultrasound on HeLa cells. Ultrasound in Med. Biol. 3, 205-219. Williams, A. R. (1973) A possible alteration in the permeability of ascites cell membranes after exposure to acoustic microstreaming. J. Cell Sci. 12, 875-885.
THE
0301-56291811020175-10502.0010 PergamonPress Ltd.
EFFECT OF 1 MHz ULTRASOUND ON THE PROLIFERATION OF SYNCHRONIZED C H I N E S E H A M S T E R V-79 C E L L S
VICTOR CIARAVINO and MORTON W. MILLER Department of Radiation Biology and Biophysics, School of Medicine and Dentistry, The University of Rochester, Rochester, NY 14642, U.S.A.
and GARY E. KAUFMAN Department of Radiology and the Franklin McLean Memorial Research Institute, The University of Chicago, Chicago, IL 60637, U.S.A. (First received 22 April 1980; and in final form 30 July 1980)
AbstraetmMitotically synchronized Chinese hamster V-79 cells were exposed to continuous wave l MHz ultrasound at a spatial peak intensity of 3 W/cm 2 at 37°C. A fraction of the cells was lysed immediately, and a second fraction was rendered nonviable. Many of these intact but nonviable cells appeared to be morphologically damaged. The remaining viable cells formed smaller colonies than did unexposed controls, apparently because of the death of one daughter cell at or shortly after the first division. There were no apparent effects on cells surviving beyond that period, and progression through the cell cycle was unperturbed. Key words: Ultrasound, Cells, Cell cycle, Membrane, Survival.
INTRODUCTION
Exposure of cultured mammalian cells to ultrasound at frequencies and intensities similar to those used in medical therapy results in the lysis of a fraction of the cell population (Clarke and Hill, 1970; Fu et al., 1980; Kaufman et al., 1977). In addition, many of the remaining intact cells are rendered nonviable as determined by vital dye exclusion and colony-forming ability (Kaufman et al., 1977; Li et al., 1977). Sonicated cells grow more slowly in monolayer culture (Kaufman and Miller, 1978) and form smaller colonies (Miller et al., 1977) than do unirradiated controls. A major site for ultrasound damage appears to be the cell membrane (Williams, 1973; Watmough et al., 1977). In the present study we examine the relationship between visible morphological damage visible by phase contrast microscopy and cell death in sonicated cells. The biological mechanisms responsible for decreased growth rate in sonicated cells are also examined. Kaufman and Miller (1978) showed that at least part of the" decreased growth rate was the result of non-viable cells being sloughed off and lost from the monolayer. However, to account for formation of smaller-than-normal colonies (Miller et al., 1977), part of the cell loss must
occur at or after the first post-sonication division, and/or there must be an increase in the duration of the cell cycle. Using synchronized cells, we examine both of these possible mechanisms. METHODS o r PROCEDURE
Cell line, medium and growth conditions A Chinese hamster V-79 cell line, originally established by Dr. M. M. Elkind, was obtained from Dr. L. J. Tolmach's laboratory (Washington University, St. Louis, MO). The cells were maintained at 37°C in a 5% CO2 water-saturated environment and grown in F-10 Ham medium supplemented with 10% calf, 10% fetal calf sera, and an antibiotic (kanamycin, 100/zg/ml). Harvesting of mitotic cells and sonication The cell population used in each experiment w/Is obtained from asynchronous, exponentially growing ,gultures grown in 490cm 2 polystyrene tissue culture roller flasks (Corning Glass Works, Corning, New York) rotating at 0.3 rpm 2-3 days before use. Cells in the mitotic phase of the cell cycle were accumulated by the addition of 0.06/xg/ml colcemid (Gibco, Grand Island, New York) for 1 hr. At that time, the cells were selected by gentle shaking of the roller
175
176
V. CIARAVINO et al.
bottle, a modified method of Highfield and Dewey (1975). The cells were then centrifuged and resuspended in fresh medium to remove the colcemid. The mitotic index of the cells at the onset of each experiment was determined to be 90-95%, which is in agreement with Highfield and Dewey (1975). Three ml aliquots of suspended cells ( 105cells/ml) were exposed to ultrasound in polystyrene tubes as described by Kaufman and Miller (1978). A l/8in, steel ball radiometer shows that the tubes are exposed to a typical bell-shaped beam with approximately a 35% attenuation of the sound field due to the front half of the polystyrene tube. Fifty percent is approximately 3 mm off center. Within the tube there appear to be standing waves as evidence by points in the tube where a steel bali radiometer shows almost no movement with changes in the power to the transducer. Elsewhere in the tube the steel ball appears to get caught in streaming eddies and exhibits an orbital motion. Hence, the physical situation within the tube is quite complex. All exposures were continuous wave (CW) at a frequency of 1.07 MHz, and at a spatial peak intensity of 3.0 W/cm 2 (37°C) as determined by a steel ball radiometer (Dunn et al., 1977). All control populations were sham-exposed.
durations of l, 2, 5 and l0 rain. After sham and ultrasound exposures, the cells were counted on a Coulter Counter Model B (Coulter Electronics, Hialeah, Florida). The percentage of intact cells was determined and aliquots of cells (200-300 cells for control plates, - 2 - 3 x l03 cells for "sonicated" plates) were placed in 100 mm diameter Petri dishes (Falcon, Oxnard, CA). Dishes were then coded before scoring. At various time intervals a dish was removed from the incubator and scored for the number of cells per colony, 100 colonies per dish were scored. For the initial time interval (2 hr), the data were derived from potential colonies which had either one or two cells (i.e. l < average value<2). The plates were then replaced in the incubator and the procedure repeated at various intervals for a period of 54 hr. There were four plates each for control and sonicated cells; in this manner one particular plate was scored a maximum of three times in the 54 hr period of any one experiment. This alleviated the problem of using one plate and repeatedly scoring the colonies, thereby decreasing the chance of cells being detached from the monolayer. The experiment was conducted three times for each exposure duration.
Daughter cell survival Cell growth After treatment, the control and sonicated cell suspensions were counted on a Coulter Counter and the cells were placed into six 25 cm 2 culture flasks (Falcon, Oxnard, CA), three of which were controls ( - 1 . 5 x 105 cells/flask). At l, 2 and 3 hr intervals after sonication, a control and a "sonicated" flask were removed from the incubator, the cells detached by agitation and the cell number was again determined. The fraction of viable cells was determined by the ratio of the number of cells excluding trypan blue in 0.4% aqueous solution to the total cell number (Phillips, 1973); one hundred cells per slide were scored. The experiment was repeated four times; all slides were scored blind. Preliminary data on cells fixed in 3:1 methanol:acetic acid or flame-killed cells indicated 100% non-viable cells with the trypan blue staining procedure.
Colony multiplicity Mitotic cells were exposed to 1MHz ultrasound at an intensity of 3 W/cm 2 for
The possibility that one of the two daughter cells may be lost at or shortly after division was tested. After sonication or sham sonication at 3 W/cm 2 for 1 min, mitotic cells were pipetted into 125 ml Erhlenmeyer flasks containing conditioned medium (conditioned medium contains equal amounts of fresh medium and medium previously used to support cell growth) and placed on a shaker at 37°C for 90 min. Carrano (1973) states that cells are almost completely released from mitosis (or more correctly, from metaphase arrest) within 45 min after conditioned medium is added. The cell suspensions were then placed on 100mm Petri dishes and scored for colony multiplicity as previously described. The experiments were repeated four times.
Incidence of polyploidy Mitotic cells were exposed to 3W/cm 2 ultrasound for 1 min. The sham and treated cells were placed on 100 mm Petri dishes and 5 mls of fresh medium added, 3 dishes each for sham and treated cells. The dishes were
The effect of 1 MHz ultrasound on the Chinese hamster V-79 cells
then placed in the incubator at 37°C. At 24 hr post-sonication 0.30 ml of colcemid (0.06 tzg/ml) was added to two of the dishes, one control and one "sonicated," for 4 hr to accumulate mitotic figures. The mitotic cells were then collected, given a 10 min treatment in hypotonic solution (1:1 F-10 medium:distilled water), and fixed in 3:1 methanol:acetic acid. Slides were prepared and Giemsa stained. The same procedure was repeated at 46 and 72 hr after sonication. The slides were then coded and the percent of cells with polyploid mitotic figures determined. The experiments were scored blind and repeated 4 times.
Autoradiographical analysis of progress into the S phase of the cell cycle after sonication Mitotically synchronized cells were exposed to 3 W/cm 2 ultrasound for 1 min. The cells were placed onto 35 mm Petri dishes and 2 ml of fresh medium containing tritiated thymidine (1.0~Ci/mM) were added. The dishes were then placed in an incubator at 37°C. At hourly intervals duplicate dishes of sham and ultrasound treated cells were removed from the incubator and fixed with 3:1 methanol:glacial acetic acid. The dishes were then treated with 2% perchloric acid (W/V) overnight in a refrigerator, rinsed with distilled water and with 95% ethanol and air dried. NTB-3 photographic emulsion (Eastman Kodak, Rochester, New York) at 45°C was poured into the dishes. The excess emulsion was poured off, and the dishes were inverted to dry for several hours. The plates were then stored in a light-tight box in the refrigerator for 1-2 weeks, and developed with D-19 (Eastman Kodak, Rochester, New York) for 2 min at 19°C. After fixation, the dishes were Giemsa stained and coded before scoring. Dishes were scored for percent 3Hnuclear labeled cells (Quastler and Sherman, 1959).
177
(1.0ttC/ml). After 30min the dishes were removed from the incubator and treated in the same manner as described in the above section. Development of autoradiographical dishes and scoring was as described above. The cell cycle was also assessed by determining the frequency of cells in mitosis after release from synchrony. Approximately 104 cells were placed onto 25cm 2 Falcon flasks and 5 ml of conditioned medium added after a 1 min sonication at 3 W/cm 2. At 6, 12, 14, 16, 18 and 20 hr post-sonication the cells were analyzed for the percent mitotic cells in the population. One hour before a designated time, 0.30 ml colcemid (0.06mg/ml; Gibco, Grand Island, New York) was placed into the flasks. After one hour the medium was removed and placed in a test tube and spun down. At that time 5 ml of 0.5% sodium citrate were placed both on the remaining attached cells and on the cell pellet in the test tube. After 30 min the cells were detached by vigorous shaking of the flask; the contents of the flask and the tube were combined, spun down and fixed in 3:1 methanol:glacial acetic acid. Slides were prepared and scored as described previously. The cell cycle experiments were repeated four times.
Cell survival
The plates used in the colony multiplicity experiments were kept in a growth incubator for 7 days, then fixed in 3:1 methanol:glacial acetic acid for 5 min, and Giemsa stained. The number of colonies on the plates was counted using a dissecting microscope. Colonies which contained fewer than fifty cells were not included in the total number of colonies. The plating efficiency of cells was determined as the number of colonies per plate divided by the number of intact cells plated. The surviving fraction was calculated by dividing the plating efficiency of sonicated cells by the plating efficiency of sham treated Analysis of progression through the 1st cell cells. The experiments were repeated four times.
cycle after sonication
Mitotically synchronized cells were exposed to 3 W/cm 2 ultrasound for 1 min. Progress through the cell cycle immediately following sonication was assessed in two ways. Cells were placed onto 35 mm Petri dishes and at 2 hr intervals dishes containing control and sonicated cells were removed from the incubator, the medium was aspirated and replaced with medium containing 3H-TdR
Analysis of morphological perturbation Within 15 min after sonication at an intensity of 3W/cm 2 for 0, 1, 2, 5 or 10min cells were plated onto clean microscope slides and viewed unstained, through a Zeiss Photomicroscope (phase contrast, 80x magnification). Slides were scored for percent aberrant cells, that is, cells which appeared morphologically damaged.
178
V. CIARAVINO et al. RESULTS
Initial cell lysis Exposure of mitotically synchronized Chinese hamster V-79 cells to 1 MHz ultrasound at an intensity of 3 W/cm 2 resulted in the immediate lysis of a fraction of the cell population. As shown in Fig. 1, the amount of cell lysis was directly correlated to the time during which cells remained in the sound field. Temperature rises do not appear to be involved in the cellular changes reported since the cells are maintained at 37°C before, during and after sonication. For a 1.0min exposure there were 83.56+_1.92 (S.E.)% cells remaining intact. This percentage decreased to 53.06-+6.71% for a 2min exposure, 39.98+-4.30% for a 5 min exposure and 7.63+-1.04% for a 10min exposure.
Cell growth The changes in cell number and viability during the first 3 hr after sonication for 1 min at 3 W/cm 2 are given in Table 1. The growth index in column 1 is the ratio of the number of cells in the flask at the indicated time to
100 : - -
--~ ~ 10
g
~ 5! (..) ~,
~
z
o
FREQUENCY 1 MHz I N T E N S I T Y 3W/cm 2 (~MEAN ± S E
~
~
~
the number of intact cells initially plated. Column 2 indicates the number of non-viable cells as determined by trypan blue dye exclusion. The growth index is then corrected for non-viable cells (column 3) by multiplying the growth index (column 1) by (1.00-nonviable cell index). The number of cells in the control flasks increased approximately 60% in the first hour as the cells divided, then remained steady for the next 2 hr. The fraction of non-viable cells was approximately 15% and remained about constant. The number of sonicated cells (and their daughters) likewise increased in the first hour by approximately the same percentage as controls, but approximately twice as many sonicated cells (i.e. - 3 0 % ) were non-viable. During the next 2hr there was a decrease in cell number, indicating some cell lysis, and an increase in the fraction of cells unable to exclude the trypan blue, indicating an increase in the number of non-viable cells detected.
Colony multiplicity Colony multiplicity data are shown in Fig.2 for an exposure duration of 1 min, and given in Table 2 for exposure durations of up to 10min. Colonies which were derived from sonicated cells were smaller (i.e. contained fewer cells) than controls. This difference appeared within the first few hours after sonication. Thereafter growth rates were comparable for controls and sonicated cells.
Daughter cell survival ~
l~
E X P O S U R E D U R A T I O N (minutes)
Fig. 1. The relationship between sonication duration and percent intact cells at an exposure of 3W/cm 2. Each point in this figure presents the mean-+S.E, of three experiments. Cell counts were determined in triplicate.
In these experiments the cultures were maintained in suspension for 90 min after sonication or sham-sonication to allow the first division to be completed before the cells were plated. Thus the colonies were started from single daughter cells. There was no
Table 1. The growth of V-79 cells after exposure to 3 W/cm 2 for 1 min. The corrected growth index of sonicated cells has been normalized to their respective controls. (Refer to Results, Cell growth)
Control
Hr. a f t e r Sonication
Growth Index
Non-Viable Cell Index
Growth Index Corrected f o r Non-Viable Cells
1 2 3
1.57 1.65 1.52
O. 14 0.16 0.13
i . 35 1.39 1.32 Normalized Values
Sonicated
i 2 3
1.56 i . 43 1.38
0.29 fh 34 0.42
1.11 O. 94 0.80
0.82 O. 68 0.59
The effect of 1 MHz ultrasound on the Chinese hamster V-79 cells
179
Table 2. Colony Multiplicity of Chinese Hamster V-79 Cells After Exposure to 3 W/cm 2 Ultrasound Exposure Duration (min)
Time After Exposure (hrs)
Colony M u l t i p l i c i t y (no. cells/colony)
Exposed
i
2
5
i0
Control
1 2 3 4 6 12 24 30 48 54
1.07 1.28 1.26 1.22 1.21 1.50 2.33 2.32 5.44 7.62
1.27 1.46 1.42 1.47 1.50 1.71 2.67 2.85 6.72 9.59
.84 .88 .89 .83 .81 .88 .87 .81 .81 .79
2 3 6 12 24 30 48 54
1.08 1.31 1.20 1.36 i . 87 2.07 3.35 4.85
i . 19 1.51 1.47 1.65 2.35 2.72 4.28 6.96
.91 .87 .82 .82 .80 .76 .76 .70
2 6 24 30 48 54
1.16 ].27 i . 86 2.20 3.70 5.66
1.27 1.54 2.46 3.05 5.25 7.12
.ql .82 .76 .72 .70 .79
2 6 24 30 48 54
i.i0 1.15 2.37 2.71 4.73 5.77
1.31 1.49 2.98 3.22 5.98 8.15
.84 .77 .80 .84 .79 .71
Analysis o[ progression
l0
..~
Ratio Exposed/Control
FREQUENCY 1 MHz INTENSITY 3 W/cm2/1 min
HOURS POST-SONtCAT~ON Fig. 2. The formation of colonies with time from single cells after sonication at 3 W/cm ~ for 1 rain (0) or sham sonication (©). Each point in the figures represents the mean for three experiments, with 100 colonies scored in each experiment.
The progression of cells through G~ and into the first S period after sonication is shown in Fig. 3. The progression of cells through the first cell cycle after sonication as shown by pulse 3H-labeling is depicted in Fig. 4. Figure 5 illustrates the mitotic index of cells for the 20hr period after sonication. There were no statistically significant differences between exposed and control populations in any of these three sets of 1oo 80. ~
60
m 40
DUM~ONs E1 rnln
subsequent difference detected in colony multiplicity between colonies derived from sonicated and sham-sonicated cells. HRS. POST-SONICATION
Incidence of polyploidy Of the 1800 cells scored there was only one polypoid observed and it appeared in a slide from sham treated cells. Thus neither the colcemid treatment nor the sonication caused a significant number of polyploid cells.
Fig. 3. The relationship between the percent 3H-labeled cells and hours post-sonication. After sonication cells were placed in continuous 3H-TdR (1.0 #Ci/mi) label. At hourly intervals plates were rinsed, fixed and the plates scored for percent 3H-labeled cells. Each point represents the mean _+S.E. of three experiments. Duplicate plates were used in each experiment (O-control, O-sonicated).
V. C1ARAVINO el al.
180
reduction (to - 2 . 5 % , or - 8 % of the control value).
Analysis of morphological perturbations Normal and aberrant-type mitotic cells are shown in Figs. 6(a, b). Figure 6(a) is a normal mitotic cell, well rounded and with a clearly 3 ] ~ 6 8 ; ,: L~ 8 2C defined cell membrane. In Fig. 6(b), the cell H O U R S POST SONICATION type illustrated is a mitotic cell but is Fig. 4. 3H-labeled cells as a function of time after soni- easily identified as an aberrant cell. The cation. Exposure of mitotic cells was at 3 W/cm2 for membrane is obviously damaged and l min. The experiments were repeated 4 times (O-concytoplasm appears to be emanating from the trol, Q-sonicated). cell; this process is characteristic of cell lysis. ~oo Approximately 21% of the control cells IMHZ were aberrant as determined by the above ~U~ATION I MIN criteria. A 1 min exposure to ultrasound increases the fraction of aberrant cells to 39%, and a 10 min exposure to 70%. An aberrant frequency corrected for the control ~ 20 frequency of aberrants (i.e. the increase in aberrant frequency from controls divided by 0 6 16 20 HOURS POST SONICATION the control fraction of nonaberrants) is given Fig. 5. Percent mitotic cells as a function of hours post in Table 3. This corrected f r e q u e n c y of abersinication. Exposures were at 3W/cm2 for 1 min. The rants is approximately 20% after a 1 min experiments were repeated 4 times (A-control, O-soniexposure to ultrasound, and increases to apcated). proximately 62% after a 10 min exposure. 1)
FREQUENCY
(~)
I MEAN . S E
12
~4
18
progression experiments, as determined by paired t tests and by analysis of covariance (p>0.05). Both the control and sonicated cells demonstrate a mitotic peak at 18 hr postultrasound treatment.
Plating efficiency The plating efficiency of control mitotic cells which were treated for l hr with 0.06 tzg/ml colcemid averaged - 3 0 % . As indicated in Table 3, a 1 min exposure to ultrasound reduced this plating efficiency by approximately 66% (to - 1 0 % ) , while a 5 or 10min exposure resulted in about a 92%
DISCUSSION
Exposure of synchronous Chinese hamster V-79 cells to 1 MHz ultrasound for 1-10 min at an intensity of 3 W / c m 2 resulted in the immediate lysis of a fraction of the cell population (Fig. 1). Microscopic examination of the remaining cells in the first few minutes after exposure revealed that many were morphologically damaged and appeared to be in the process of losing their physical integrity; presumably most, if not all, of these damaged cells subsequently lysed. These results are consistent with previous studies. Williams (1973) exposed mouse Ehrlich
Table 3. Platingefficiencyand aberrant cells after exposure to 3 W/cm2ultrasound. Values given are mean _+ standard error of the mean Duration of Exposure (min) i 2 5 10
•
Reduction in Plating Efficiency *(%) 66 • R6 ~ 92 i 91 *
3 4 3 4
Increase in Frequency of Aberrants f(%) 22.5 36.0 50.8 62.3
• ~ * *
2.6 2.3 6.4 I0.I
I - ( P / P o ) , where P is p l a t i n g e f f i c i e n c y a f t e r a given exposure, and Po is p l a t i n g e f f i c i e n c y f o r c o n t r o l s . Based on three experiments, four plates per experiment at each exposure d u r a t i o n .
(X-Xo)/(1-Xo), where X is the frequency of aberrant cells after a given exposure, and Xo is the frequency of aberrants for controls. Based on four experiments, 100 cells per experiment at each exposure duration.
The effect of 1 MHz ultrasound on the Chinese hamster V-79 cells
la)
(b)
Fig. 6. Photomicrographs of mitotic cells within 15 min after sonication or sham-sonication. Figure 6(a) shows a normal miotic cell. Figure 6(b) shows an aberrant cell. Photomicrographs were taken on a Zeiss photomicroscope at an 80X magnification using Kodak high contrast copy film. ( - 900X).
181
The effect of 1 MHz ultrasound on the Chinese hamster V-79 cells ascites carcinoma cells to a small-scale acoustic microstreaming field generated around a transversely oscillating wire driven at 20 kHz, and observed cells with large areas of plasma membrane torn off, but with the cytoplasm and organelles still attached to each other. Watmough et al. (1977) conducted electron microscopic examinations of HeLa cells after treatment with 0.75 MHz ultrasound at spatial peak intensities of 1W/cm ~. Damage was observed to the nuclear and mitochondrial membrane systems as well as to the plasmalemmal and endoplasmic reticulum. The morphologically damaged cells account for a significant fraction, but not all, of the loss in viability in those sonicated cells which are not lysed immediately (Table 3). This result is not surprising, since Kaufman and Miller (1978) observed that some cells rendered nonviable by ultrasound can remain floating in the medium for several days without lysing. The overall loss of viability observed in the present study (including morphologically damaged cells and those not so damaged) is consistent with that reported previously (Kaufman and Miller, 1978; Fu et al., 1980). As might be expected, with longer exposures the morphological aberrants account for a larger fraction of the nonviable cells, reflecting an overall increase in damage to the cell population. The effect of ultrasound on cell growth was examined by measuring changes in total cell number (or number of viable cells as determined by vital dye exclusion), and by measuring the average number of cells per colony (colony multiplicity). Both types of experiments indicated a reduction in growth in the sonicated cells. These results are similar to those reported previously for asynchronous cells (Miller et al., 1977; Kaufman and Miller, 1978). As described in the introduction, two mechanisms could account for this decreased growth--an increase in the duration of the cell cycle, and/or a loss of cells from the proliferating population. Progression through the cell cycle was assessed by chronic and pulse labelling experiments (Figs. 3 and 4, respectively) and by mitotic index determinations (Fig. 5). No difference between exposed and control cells was observed with respect to progression. Hence there was little or no effect on the duration of the cell cycle.
183
However, both the growth experiments (i.e. change in total cell number) and the colony multiplicity experiments indicated a loss of cells from the proliferating population. The growth experiments (Table 1) indicate that after a 1 min exposure most of the cells which were not immediately lysed proceeded through the first division and were still intact 1 hr after exposure, although a number of the daughter cells were nonviable as determined by trypan blue exclusion. During the next 2 hr there was a decrease in cell number and a • lecrease in the fraction of cells able to exclude the vital dye. Thus in the period from 1-3 hr after exposure some daughter cells were lysed, while other lethally damaged daughter cells lost the ability to exclude trypan blue. The colony multiplicity experiments give a similar picture of cell loss within the first few hours after exposure, although the effects appear more pronounced within the first hour after exposure (Fig. 2 and Table 2). This may mean that many of the intact but lethally damaged daughter cells fail to reattach to the plates after division. There appears to be a slight drop in colony multiplicity between 2 and 6 hr after sonication, for cells sonicated for 1 min, which may represent a second population of lethally damaged daughter cells which initially reattach after division, but are later sloughed off into the medium or lyse. This would be similar to the results of Kaufman and Miller (1978), who observed that, in asynchronous V-79 cells, sonication resulted in a large population of nonviable cells which initially reattached but were later sloughed off. However, the changes in colony multiplicity during this period are small and may not be significant. Beyond the first several hours after exposure there were no apparent effects on cell growth, with the ratio of colony multiplicity between sonicated and control cells remaining nearly constant. Hence the effects of ultrasound on colony size appear to be due entirely to colonies in which one daughter cell was lost at or shortly after division, while the effects on growth (total cell number) would also reflect colonies which were never formed since both daughters were lost. In the case of asynchronous cells there may also be significant loss of cells prior to the first division, which would contribute to effects on growth (Kaufman and Miller, 1978) but not to decreased colony size (Miller et al., 1977).
184
V. CIARAVINOet al.
Acknowledgements--The authors are indebted to Ms. Valarie Ferro for excellent technical assistance during the course of this research. This paper is based on work performed under contract no. DE-AC02-76EV03490 with the U.S. Department of Energy at The University of Rochester, Department of Radiation Biology and Biophysics and has been assigned Report No. UR-3490-1777. This work was also partially supported by USPHS Grants GM22680, FD00694 and CA25785. REFERENCES
Carrano, A. V. (1973) Chromosome aberrations and radiation-induced cell death. I. Transmission and survival parameters of aberrations. Mutation Research 17,341-353. Clarke, P. R. and Hill, C. (1970) Physical and chemical aspects of ultrasonic disruption of cells. J. Acoust. Soc. Am. 50, 649. Dunn, F., Auerbach, A. J. and O'Brien, Jr., W. D. (1977). A primary method for the determination of ultrasonic intensity with elastic sphere radiometer. Acustica 38, 58-61, Fu, Y-K., Miller, M. W., Kaufman, G. E., Lange, C. S. and Grifliths, T. D. (1980) Ultrasound lethality to synchronous and asynchronous Chinese hamster V-79 cells. Ultrasound in Med. and Biol 6, 39-46. Highfield, D. P. and Dewey, W. C. (1975) Use of mitotic selection procedure for cell cycle analysis: Emphasis on radiation-induced mitotic delay. In Methods in Cell
Biology (Edited by Prescott, D. M.), Vol. IX, pp. 85-101. Academic Press, New York. Kaufman, G. E. and Miller, M. W. (1978) Growth retardation in Chinese hamster V-79 cells exposed to l MHz ultrasound. Ultrasound in Med. and Biol. 4, 139-144. Kaufman, G. E., Miller, M. W., Grittiths, T. D. and Ciaravino, V. (1977) Lysis and viability of cultured mammalian cells exposed to l MHz ultrasound. Ultrasound Med. Biol. 3, 21-25. Li, G. C., Hahn, G. M. and Tolmach, L. T. (1977) Cellular inactivation by ultrasound. Nature 267, 163165. Miller, M. W., Ciaravino, V. and Kaufman, G. E. (1977) Colony size and giant cell formation from mammalian cells exposed to 1 MHz ultrasound. Radiat. Res. 71, 628-634. Phillips, H. J. (1973) Dye exclusion tests for cell viability, In Tissue Culture, Methods and Applications (Edited by Kruse, F. F. and Patterson, M. K.), pp. 406-408. Academic Press, New York. Quastler, H. and Sherman, F. G. (1959) Cell population kinetics in the intestinal epithelium of the mouse. Exptl. Cell Res. 17, 420-438. Watmough, D. J., Dendy, P. P., Eastwood, L. M., Gregory, D. W. and Gordon, F. C. A. (1977) The biophysical effects of therapeutic ultrasound on HeLa cells. Ultrasound in Med. Biol. 3, 205-219. Williams, A. R. (1973) A possible alteration in the permeability of ascites cell membranes after exposure to acoustic microstreaming. J. Cell Sci. 12, 875-885.